Abstract/Summary Pancreatic ductal adenocarcinoma (PDAC) has a high incidence of tumor heterogeneity with many mutations occurring at low frequencies (i.e. <10%)66. Accordingly, the effect of inhibitors on a broader signaling network of enzymes needs to be monitored at a single cell level to study the intrinsic mechanisms of resistance in tumor cells7. Although single cell sequencing is used to map signaling pathways using gene expression, transcript abundance and gene sequence cannot reliably infer the actual enzyme or pathway signaling within a cell. However, chemical cytometry uses sensitive analytical platforms, like capillary zone electrophoresis (CE), to investigate directly enzyme activity in single cells49. My proposal is to combine the CE chemical cytometry platform with RNA-sequencing to measure the transcriptome and enzyme activity of single cells. I will use this to study the resistance mechanisms of tumor populations in response to targeted therapies for PDAC. In aim 1, I will use opposing mobilities of peptides and nucleic acids to develop a method to collect nucleic acids during a CE enzyme activity assay. Preliminary results demonstrate that DNA and RNA standards move in the opposite direction from peptide reporters in the capillary under and electric field. I will optimize the buffers used in my CE method to collect nucleic acids quantitatively during an Akt enzyme reporter separation from the same biological sample. In aim 2, I will apply this collection method to single cells using the Allbritton lab?s automated single cell CE platform. An iterative design process will be used to redesign the sample cassette to include a new nucleic acid collection region. I?ll demonstrate its feasibility using quantitative polymerase chain reaction (q-PCR) on collected nucleic acids from single cells. In aim 3, I will analyze gene expression of cells resistant to therapies targeting ERK and PI3K-Akt. Tumor cells that are resistant to ERK inhibition show increased PI3K-Akt activity but remain resistant when PI3K is co-inhibited13. I hypothesize that increased activators of PI3K and decreased regulators of Akt are responsible for this aberrant activity and provide heterogenous mechanisms for intrinsic resistance. Using this combined platform, I will measure Akt activity after PI3K inhibition to identify resistant cells and compare their gene expression to identify patterns in resistant cells.